Sven Lindstedt

Treatment of hereditary tyrosinaemia type I by inhibition of 4-hydroxyphenylpyruvate dioxygenase.

Liver transplantation is the only effective treatment for hereditary tyrosinaemia type I (McKusick 276700). We have treated one acute and four subacute-chronic cases with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), a potent inhibitor of 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27), to prevent the formation of maleylacetoacetate and fumarylacetoacetate and their saturated derivatives. The oral daily dose was 0.1-0.6 mg/kg. The excretion of succinylacetoacetate and succinylacetone decreased from 15-103 mmol/mol creatinine to the detection limit or slightly above (ie, to 20-150 mumol/mol creatinine). The concentration of succinylacetone in plasma decreased from 5.8-43 mumol/l to the detection limit (0.1 mumol/l) over 2-5 months of treatment. The almost complete inhibition of porphobilinogen synthase in erythrocytes was abolished and the excretion of 5-aminolevulinate decreased to within or slightly above the reference range. The concentration of alpha-fetoprotein decreased in four patients to 1.3-7.5% of initially high values over 6-8 months. Improved liver function was reflected by normal concentrations of prothrombin complex and in decreased activities of alkaline phosphatase and gamma-glutamyltransferase in serum. Computed tomography revealed regression of hepatic abnormalities in three patients. One patient developed rickets 6 months before treatment and had excreted high concentrations of markers of tubular dysfunction--after 3 weeks of treatment, this excretion had disappeared. No side-effects were encountered. Inhibition of 4-hydroxyphenylpyruvate dioxygenase may prevent the development of liver cirrhosis and abolish or diminish the risk of liver cancer. Normalisation of porphyrin synthesis will eliminate the risk of porphyric crises. This type of treatment may thus offer an alternative to liver transplantation in hereditary tyrosinaemia.

A method for the determination of carnitine in the picomole range

Abstract A method is described for the determination of L -carnitine in the range 20–2000 pmoles. Incubation of L -carnitine with [1-14C]acetyl-coenzyme A and acetyl-CoA: carnitine O-acetyltransferase (EC 2.3.1.7) yields labeled acetylcarnitine. The formed acetylcarnitine is separated from labeled acetyl-coenzyme A by passing the reaction mixture through a column of the anion exchange resin Dowex 2-X8 and its isotope content determined in a liquid scintillation counter. Short-chain acyl derivatives of L -carnitine are substrates for the enzyme and will also be determined through exchange in the enzymic reaction. In 33 samples of human blood plasma the concentration of carnitine (+short-chain acyl carnitine) was 51 ± 10.5 μmoles/l (mean ± S.D.). In biopsies of human skeletal muscle obtained at surgery the concentration was 12.8 ± 2.9 μmoles/g dry weight, in mouse skeletal muscle 1.73 ± 0.27 μmoles/g dry weight, and in mouse heart 4.66 ± 0.73 μmoles/g dry weight. The precision of the method expressed as relative standard deviation of duplicate analyses was 2–5%. The recovery of carnitine added to plasma or to perchloric acid extracts of muscle was 97 ± 12.5% and 103 ± 5.9%, respectively.

Pharmacological correction of neonatal lethal hepatic dysfunction in a murine model of hereditary tyrosinaemia type I

Hereditary tyrosinaemia type I, a severe autosomal recessive metabolic disease, affects the liver and kidneys and is caused by deficiency of fumarylacetoacetate hydrolase (FAH). Mice homozygous for a FAH gene disruption have a neonatal lethal phenotype caused by liver dysfunction and do not represent an adequate model of the human disease. Here we demonstrate that treatment of affected animals with 2–(2–nitro–4–trifluoro–methylbenzyol)–1,3–cyclohexanedione abolished neonatal lethality, corrected liver function and partially normalized the altered expression pattern of hepatic mRNAs. The prolonged lifespan of affected animals resulted in a phenotype analogous to human tyrosinaemia type I including hepatocellular carcinoma. The adult FAH−/− mouse will serve as useful model for studies of the pathophysiology and treatment of hereditary tyrosinaemia type I as well as hepatic cancer.

Tyrosinaemia type I and NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione)

In tyrosinaemia type I (McKusick 276700), fatal liver disease results either because of liver failure during infancy or early childhood or because of development of hepatocellular carcinoma during childhood or adolescence. This is caused by toxic metabolites which accumulate because of deficiency of fumarylacetoacetase, the last enzyme in the tyrosine catabolic pathway. NTBC is a potent inhibitor of 4-hydroxyphenylpyruvate dioxygenase and has been shown to efficiently prevent tyrosine degradation, and production of succinylacetone, in patients with tyrosinaemia. Since the first trial of NTBC treatment for tyrosinaemia type I in 1991, over 220 patients have been treated by the drug using a protocol which includes regular follow-up with reports of clinical and laboratory investigations to the study centre in Gothenburg, where additional analysis of critical variables is done on regularly collected samples. The course of the disease in patients with acute tyrosinaemia has changed dramatically. Only 10% of the patients have not clinically responded to NTBC treatment. In half of these patients, successful liver transplantation has been performed which has further reduced the mortality rate during infancy to 5%. The international NTBC study has now been going for 5 years and data have emerged that indicate a decreased risk for early development of hepatocellular carcinoma in patients who started treatment at an early age. There are now 101 patients aged 2–8 years who have started NTBC treatment before 2 years of age, and no cancer has developed after 2 years of age among these patients. However, there is no safe age with respect to occurrence of liver cancer, which has been recognized at diagnosis at 1 year of age in one patient and after a few months of treatment in an infant who was given NTBC at 5 months of age.

CARNITINE DEFICIENCY INDUCED BY PIVAMPICILLIN AND PIVMECILLINAM THERAPY

Short-term administration of pivampicillin and pivmecillinam resulted in a reduction of serum carnitine concentration and an increase in excretion of acylcarnitine in urine. These changes persisted for more than ten days after cessation of therapy. In seven girls on long-term treatment with a mixture of pivampicillin and pivmecillinam the mean total serum carnitine concentration fell to 15% (7-27%) of pretreatment values. The acylcarnitine fraction was 11-57% of total carnitine, compared with less than 2% before treatment. Muscle carnitine concentrations in two girls treated with the antibiotics for 22 and 30 months were only 10% of the mean reference value. These concentrations in serum and muscle are in the range encountered in patients with carnitine deficiencies of other aetiologies in which life-threatening metabolic crises may arise. The risk of adverse effects from prodrugs that give rise to pivalic acid should be seriously considered, particularly in patients under metabolic stress.

NONTRANSPLANT TREATMENT OF TYROSINEMIA

NTBC treatment has greatly improved the survival of patients with acute tyrosinemia and has reduced the need for liver transplantation during early childhood. In patients in whom treatment with NTBC was started early in life, 2 cases (1%) of HCC have occurred during the first year of treatment, but no further cases have occurred among these patients, who have been followed for up to 9 years. In patients with late start of NTBC treatment, there is a considerable risk for liver malignancy. The risk for malignancy in this group of patients must be evaluated on an individual basis, taking into account the phenotype and clinical status of the patient. Porphyric crises are not seen in patients who comply with the medication regimen. NTBC is a well-tolerated drug with few adverse effects.

Concentration of carnitine in human muscle tissue

Abstract The concentration of acid-soluble carnitine has been determined in human muscle tissue and in plasma. One group of subjects consisted of patients mainly suffering from cholecystolithiasis or varicose veins. Biopsies were surgically obtained under general anesthesia. The other group consisted of normal subjects in which the biopsies were obtained by a percutaneous technique. We could not demonstrate a significant change in concentration of carnitine along a muscle fiber bundle or a difference in the concentration between specimens from superficial and deep levels. The rectus abdominis muscle had a lower carnitine concentration than the muscles of the leg. The median values were 13.7 μmoles/g dry weight (range 7.7–32.5) and 17.9 μmoles/g dry weight (range 6.5–24.1), respectively. Men had slightly higher concentrations in the rectus adbominis muscle than women. Median values were 15.4 μmoles/g dry weight (range 7.7–32.5) and 12.9 μmoles/g dry weight (range 9.8–17.7), respectively. Two subjects from whom biopsies were obtained with an interval of 91 and 42 days showed nearly the same values on both occasions. No correlation was found between age and carnitine concentration in muscle tissue. The carnitine concentration in two biopsies of human heart muscle was 4.2 μmoles/g dry weight and 4.8 μmoles/g dry weight, respectively. The median value of plasma carnitine concentration was 46.2 μmoles/1 (range 17.9–74.7). The concentration of carnitine in plasma was not correlated with the concentration of carnitine in muscle tissue.

Cytotoxic treatment of adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare, aggressive tumor that is often detected in an advanced stage. Medical treatment with the adrenotoxic drug mitotane has been used for decades, but critical prospective trials on its role in residual disease or as an adjuvant agent after surgical resection are still lacking. The concept of a critical threshold plasma level of the drug must be confirmed in controlled studies. Because individual responsiveness cannot be predicted, the use mitotane is still advised for nonresectable disease. In case of Cortisol or other steroid overproduction, several drugs (e.g., ketoconazole or aminoglutethimide) may be used. Chemotherapy with single agents (e.g., doxorubicin or cisplatin) have been disappointing, with low response rates (< 30%) and a short response duration. Part of this refractoriness may be explained by the fact that ACC tumors express the multidrug-resistance gene MDR-1. Chemotherapy with multiple agents has been tested in smaller series and has resulted in significant side effects. The best results were achieved by the combination of etoposide, doxorubicin, and cisplatin associated with mitotane, achieving a response rate of 54%, including individual complete responses. To be able to make progress in treating advanced ACC disease, adjuvant multicenter trials must be encouraged. When mitotane-based therapies are used, monitored drug levels are mandatory.RésuméLe cancer de la corticosurrénale (CCS) est une tumeur rare mais agressive, détectée souvent à un stade avancé. Le traitement médical par le mitotane, une drogue adrénotoxique, est utilisée depuis plusieurs décennies, mais, on manque d’essais prospectifs critiques sur son rôle dans la maladie résiduelle ou comme agent adjuvant après résection chirurgicale. Le concept d’un niveau plasmatique dont le seuil critique doit être confirmé par des études contrôlées. Puisqu’on ne peut prédire la réponse individuelle, le mitotane est toujours conseillé dans les maladies nonréséquables. En cas d’hyperproduction de Cortisol, ou d’autres Steroides, d’autres drogues, comme par exemple, le cétoconazole ou l’aminogluthétimide peuvent être utilisées. La monochimiothérapie, avec par exemple la doxorubicine et le cisplatine, est décevante avec un taux de réponse bas (<30%) et une durée de réponse courte. Une partie de cette non-réonse peut être expliquée par le fait que les CCS expriment un gène de résistance multidrogues, le MDR-1. La plurichimiothérapie a été testée dans de plus petites séries avec des effets secondaires importants. Les meilleurs résultats ont été avec la combinaison d’étoposide, de doxorubicine et de cisplatine associés au mitotane: le taux de réponse a été de 54%, avec quelques réponses individuelles complètes. Pour faire des progrès dans le CCS avancé, il faut encourager des essais multicentriques de traitement adjuvant. En cas de thérapie utilisant le mitotane, il faut obligatoirement monitorer le taux des drogues.ResumenEl carcinoma adrenocortical (CAC) es un tumor agresivo poco frecuente que comûnmente se détecta cuando ya esta en etapas avanzadas de su desarrollo. Desde hace décadas se practica tratamiento médico con una droga adrenotöxica, el mitotane, pero no se dispone de ensayos clînicos prospectivos que soporten su valor en enfermedad residual o como adyuvante de la resection quirürgica. También se hace necesario confirmar el concepto de nivelés criticos de la droga mediante estudios controlados. Puesto que no es posible predecir la respuesta individual, todavia se aconseja el uso del mitotane en casos de enfermedad no resecable. Cuando hay superproduction de Cortisol o de otros esteroides, se pueden utilizar algunas drogas como el Ketoconazol o la aminoglutetimida. La quimioterapia con agentes ûnicos, por ejemplo doxorubicina y el cisplatino, ha sido decepcionante, por las bajas tasas (<30%) y la corta duration de la respuesta. Parte de tal situation refractaria puede explicarse por el hecho de que los CAC expresan el gen MDR-1 de resistencia multidroga. La quimioterapia con agentes multiples ha sido ensayada en series mas pequenas, con efectos secundarios significatives. Los mejores resultados se logran con la combination de etopösido, doxorubicina y cisplatino asociada con mitotane, alcanzando una tasa de respuesta del 54%, incluyendo respuestas complétas. Para lograr progreso en el manejo del CAC avanzado se debe estimular la realization de ensayos multi-institucionales. La monitoria de los nivelés de droga son obligatorios cuando se usan terapias con base en mitotane.

Effect of nitisinone (NTBC) treatment on the clinical course of hepatorenal tyrosinemia in Québec.

BACKGROUND Hepatorenal tyrosinemia (HT1, fumarylacetoacetate hydrolase deficiency, MIM 276700) can cause severe hepatic, renal and peripheral nerve damage. In Québec, HT1 is frequent and neonatal HT1 screening is practiced. Nitisinone (NTBC, Orfadin ®) inhibits tyrosine degradation prior to the formation of toxic metabolites like succinylacetone and has been offered to HT1 patients in Québec since 1994. METHODS We recorded the clinical course of 78 Québec HT1 patients born between 1984 and 2004. There were three groups: those who never received nitisinone (28 patients), those who were first treated after 1 month of age (26 patients) and those treated before 1 month (24 patients). Retrospective chart review was performed for events before 1994, when nitisinone treatment began, and prospective data collection thereafter. FINDINGS No hospitalizations for acute complications of HT1 occurred during 5731 months of nitisinone treatment, versus 184 during 1312 months without treatment (p<0.001). Liver transplantation was performed in 20 non-nitisinone-treated patients (71%) at a median age of 26 months, versus 7 late-treated patients (26%, p<0.001), and no early-treated patient (p<0.001). No early-treated patient has developed detectable liver disease after more than 5 years. Ten deaths occurred in non-nitisinone treated patients versus two in treated patients (p<0.01). Both of the latter deaths were from complications of transplantation unrelated to HT1. One probable nitisinone-related event occurred, transient corneal crystals with photophobia. INTERPRETATION Nitisinone treatment abolishes the acute complications of HT1. Some patients with established liver disease before nitisinone treatment eventually require hepatic transplantation. Patients who receive nitisinone treatment before 1 month had no detectable liver disease after more than 5 years.

Mitochondrial ATP-Synthase Deficiency in a Child with 3-Methylglutaconic Aciduria

ABSTRACT: We report the finding of mitochondrial ATP-synthase deficiency in a child with persistent 3-methylglutaconic aciduria. The child presented in the neonatal period with severe lactic acidosis, which was controlled by Na-HCO3 and glucose infusions. During the 1st y of life, there were several episodes of lactic acidosis precipitated by infections or prolonged intervals between meals. The excretion of lactate in urine was variable, but there was a persistent high excretion of 3-methylglutaconic acid. The activity of 3-methylglutaconyl-CoA hydratase in fibroblasts was normal. The child had a hypertrophic cardiomyopathy and magnetic resonance images revealed hypoplasia of corpus callosum. The gross motor and mental development was retarded, but there were no other neurologic signs. Investigation of muscle mitochondrial function at 1 y of age revealed a severe mitochondrial ATP-synthase deficiency (oligomycin-sensitive, dinitrophenol-stimulated Mg2+ ATPase activity: 27 nmol × min-1 × (mg protein)-1, control range 223–673 nmol × min-1 × (mg protein)-1. The mitochondrial respiratory rate was low and tightly coupled. The respiratory rate was normalized by the addition of an uncoupler. Low Mg2+ ATPase activity was also demonstrated by histochemical methods. Morphologic examination revealed ultrastructural abnormalities of mitochondria. There was no deletion of mitochondrial DNA. The sequences of the ATP synthase subunit genes of mitochondrial DNA were in accordance with published normal sequences.